Polymeric nanogels with degradable backbones and from gras components, and compositions and methods thereof

ABSTRACT

The invention generally relates to novel polymers and polymeric nanogels having biodegradable polymeric backbones, and compositions and methods of preparation and use thereof, for example, as guest-host polymer nano-assemblies and nano-delivery vehicles, which offer utilities in diverse fields including drug delivery, diagnostics and specialty materials.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/047,878, filed Sep. 9, 2014, the entire content of which isincorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

The United States Government has certain rights to the inventionpursuant to Grant No. GM-065255 awarded by the National Institutes ofHealth and to Grant No. W911NF-13-1-0187 awarded by the U. S. ArmyResearch Office.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to polymers and polymer-basednano-structures. More particularly, the invention relates to novelpolymers and polymeric nanogels having biodegradable polymericbackbones, and compositions and methods of preparation and use thereof,for example, as guest-host polymer nano-assemblies and nano-deliveryvehicles, which offer utilities in diverse fields including drugdelivery, diagnostics and specialty materials.

BACKGROUND OF THE INVENTION

Recently, nanoparticles have played an increasingly significant role indiverse fields such as microelectronics, multiphase catalysis, sensingand therapeutics. (Nanoparticles: From Theory to Application; Schmid,Ed.; Wiley-VCH: Essen, 2004; Zhang, et al. Self-AssembledNanostructures; Nanostructure Science and Technology Series; Springer:2002; Nanoparticles: Building Blocks for Nanotechnology; Rotello, Ed.;Springer: 2003; Daniel, et al. 2004 Chem. Rev. 104, 293.) The ability toencapsulate and release guest molecules within the nanoparticle interioris required for applications such as sensing and therapeutics. For manyapplications, facile modulation of the nanoparticle surface is alsoimportant in order to obtain appropriate interfacial properties.

Amphiphilic molecules readily self-assemble into nanoassemblies, such asmicelles and liposomes, which can encapsulate guest molecules withintheir interior spaces. (Harada, et al. 2006 Progress in Poly. Sci. 31,949-982; O'Reilly, et al. 2006 Chem. Soc. Rev. 35, 1068-1083; Zhu, etal. 2012 J. Mat. Chem. 22, 7667-7671; Owen, et al. 2012 Nano Today 7,53-65; Sawant, et al. 2010 Soft Matter 6, 4026-4044; Micheli, et al.2012 Recent Patents on CNS Drug Discovery 7, 71-86.)

A major challenge remains in developing polymeric nanogels wherein thebackbones are fully degradable. In particular, methodologies are highlydesired that allow significant control over the degradation of polymerbackbones to small molecules that are generally regarded as safe (GRAS).This challenge is magnified due to the need to maintain ahydrophilic-lipophilic balance that is necessary for retention of thefidelity of the assembly while allowing efficient surfacefunctionalization.

With regard to the development of drug delivery systems, significantattention has been paid to cancer therapy because of the severity andoften-fatal nature of the disease. (Siegel, et al. 2014 CA-Cancer J.Clin. 64, 9-29.) Nanocarriers have emerged as a superior class of drugdelivery system as they can exploit the leaky vasculature of tumortissues for selective uptake. (Danhier, et al. 2010 J. ControlledRelease. 148, 135-146; Maeda, et al. 2000 J. Controlled Release. 65,271-284; Matsumura, et al. 1986 Cancer Res. 46, 6387-6392; Davis, et al.2008 Nat. Rev. Drug Discov. 7, 771-782; Baban, et al. 1998 Adv. DrugDeliv. Rev. 34, 109-119; Duncan 2003 Nat. Rev. Drug Discov. 2, 347-360;Gillies, et al. 2005 Drug Discovery Today 10, 35-43; Peer, et al. 2007Nat. Nanotechnol. 2, 751-760; Haag 2004 Angew. Chem. Int. Ed. 43,278-282; Allen, et al. 2004 Scienc. 303, 1818-1822.)

Amongst the nanocarriers that are being developed for this purpose,polymeric micelles have attracted particular attention as thesenanoassemblies can noncovalently encapsulate the hydrophobic drugmolecules in aqueous conditions. (Kale, et al. 2009 Langmuir 25,9660-9670; Koo, et al. 2005 Nanomedicine: NBM 1, 193-212; Liu, et al.2009 Macromolecules 42, 3-13; Kataoka, et al. 2001 Adv. Drug DeliveryRev. 47, 113-131; Savic, et al. 2003 Science 300, 615-618; Torchilin2001 J. Controlled Release 73, 137-172; Yin, et al. 2008 J. ControlledRelease 131, 2-4; Kwon, et al. 1995 Adv. Drug Delivery Rev. 16, 295-309;Li, et al. 2014 Chem. Commun. 50, 13417-13432; Jeong, et al. 1997 Nature388, 860-862; Kwon, et al. 1996 Adv. Drug Delivery Rev. 21, 107-116;Gref, et al. 1994 Science 263, 1600-1603.) Although polymer micellesshow great promise in many cases, these assemblies face a generalconundrum with respect to drug loading and encapsulation stability.

For high encapsulation stability, it is necessary that the hydrophobicpart of the micellar assembly is glassy so as to keep the guestmolecules from leaking into the bulk. On the other hand, if the interiorof the assembly is glassy, loading the drug molecules become an issue.The successful utility of polymer micelles in the drug delivery area hasdemonstrated that ‘sweet spots’ can indeed be identified to developuseful nanocarriers. A complementary approach that can offer a viablesolution to this issue involves chemically crosslinked polymericassemblies, where the loading can occur when the assemblies are ratherlose and the encapsulation stability is achieved due to thecrosslinking-induced incarceration of the drug molecules. (Ryu, et al.2010 J. Am. Chem. Soc. 132, 8246-8247; Oh, et al. 2008 Prog. Polym. Sci.33, 448-477; Molla, et al. 2014 Biomacromolecules 15, 4046-4053;Rossler, et al. 2012 Adv. Drug Delivery Rev. 64, 270-279; Peppas, et al.2000 Eur. J. Pharm. Biopharm. 50, 27-46.) Such a crosslinking strategyalso offers the opportunity to program the assemblies to uncrosslink andrelease its contents only in the presence of a specific stimulus.(Ganta, et al. 2008 J. Controlled Release 126, 187-204; Shenoy, et al.2005 Pharm. Res. 22, 2107-2114; Shenoy, et al. 2005 Mol. Pharmacol. 2,357-366; Kommareddy, et al. 2005 Bioconjug. Chem. 16, 1423-1432; Meyer,et al. 2001 J. Control. Release. 74, 213-224; Saito, et al. 2003 Adv.Drug Deliv. 55, 199-215; Arrueboa, et al. 2007 Nano Today 2, 22-32; Ito,et al. 2005 J. Biosci. Bioeng. 100, 1-11; Rapoport, et al. 2002 DrugDeliv. Syst. Sci. 2, 37-46; Gao, et al. 2005 J. Control. Release 102,203-221; Rapoport 2007 Prog. Polym. Sci. 32, 962-990.)

As safety of the drug carriers is of utmost importance, it is criticalthat a carrier is biocompatible. (Matsumura, et al. 2009 Cancer Sci.100, 572-579; Kim, et al. 2004 Clin. Cancer Res. 10, 3708; Matsumura, etal. 2004 Br. J. Cancer. 91, 1775-1781; Hamaguchi, et al. 2010 Clin.Cancer Res. 16, 5058-5066; Katti, et al. 2002 Adv. Drug Delivery Rev.54, 933-961; Ulery, et al. 2011 J. Polym. Sci., Part B: Polym. Phys. 49,832-864; Friess Eur. 1998 J. Pharm. Biopharm. 45, 113-136; Middleton, etal. 2000 Biomaterials 21, 2335-2346; Deng, et al. 2012 Nano Today 7,467-480.)

Thus, there is a continued need for novel approach to developbiocompatible scaffolds is to design the components of the assembly suchthat they are biodegradable and that the degradation products arenon-toxic.

SUMMARY OF THE INVENTION

The invention provides novel polymers and polymeric nanogels havingbiodegradable polymeric backbones, and compositions and methods ofpreparation and use thereof, for example, as guest-host polymernano-assemblies and nano-delivery vehicles, which offer utilities indiverse fields including drug delivery, diagnostics and specialtymaterials.

The invention provides stimuli responsive polymeric nanogels, whereinthe polymer is designed such that the degraded products are composed ofmolecules that are generally accepted to be biocompatible, moreparticularly, molecules that are GRAS (generally regarded as safe).(http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/)

Drug delivery systems based on biocompatible molecules that not onlyencapsulate a hydrophobic drug molecule, but also release it in responseto a specific trigger are of utmost importance for therapeutic andbiomedical applications.

In one aspect, the biodegradable polymers of the invention include apolymeric backbone selected from backbones of polyamides, polyesters andpolycarbonates.

In another aspect, the biodegradable polymeric nanogel of the inventionis designed to release encapsulated guests (e.g., drug or diagnosticpayloads) in response to defined biologically, physically or chemicallyrelevant stimuli (e.g., glutathione concentration inside the cells, achange in pH, redox reagent, redox potential, ionic strength, enzymaticactivity, protein concentration, light, heat, or mechanical stress).

In yet another aspect, the nano-assembly of the invention includes ahost crosslinked polymer network having a biodegradable polymericbackbone, and is typically surface functionalized with one or morefunctional groups to allow modifiable surface functionalities (e.g., tointroduce targeting capabilities); and one or more guest molecularcargos (e.g., non-covalently encapsulated in the host crosslinkedpolymer network). The host crosslinked polymer network is addressable bya biological, physical or chemical intervention (e.g., glutathioneconcentration inside the cells, a change in pH, redox reagent, redoxpotential, ionic strength, enzymatic activity, protein concentration,light, heat, or mechanical stress) resulting in partial or completedegradation and/or descrosslinking of the host polymer network andrelease of the guest molecular cargo from the nano-assembly.

In yet another aspect, the biodegradable polymers and nano-assemblies ofthe invention are biodegradable into molecules that are generallyregarded as safe (GRAS).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Scheme 1. A schematic illustration of an exemplary embodiment ofthe biodegradable polymer nanogel delivery system.

FIG. 2. Exemplary embodiment of a biodegradable polycarbonate backbone.

FIG. 3. Exemplary embodiment of monomers that may be used to formbiodegradable polycarbonates.

FIG. 4. Exemplary embodiments of aspartic and glutamic acid-containingself-crosslinked polymer nanogels.

FIG. 5. Exemplary embodiments of serine and threonine-containingself-crosslinked polyester nanogels.

FIG. 6. Exemplary embodiments of serine and threonine-containingself-crosslinked polyester nanogels.

FIG. 7. Exemplary embodiments of lysine-containing self-crosslinkedpolymeric nanogels, serine and threonine-containing self-crosslinkedpolyamide, and lysine-containing copolymer nanogels.

FIG. 8. Scheme 2. Top: Schematic representation of hydrophobic guestencapsulation, followed by redox responsive release and degradation byenzymes; Bottom: Chemical structure of the nanogel precursor polymer.

FIG. 9. Scheme 3. Synthetic scheme for the nanogel precursor polymer andnanogel

FIG. 10. Self-assembly of polymeric aggregates as seen by a) hydrophobicguest encapsulation b) dynamic light scattering, c) transmissionelectron microscopy (scale 500 nm)

FIG. 11. (a) Absorption spectra of pyridothione in UV-vis. Pyridothione,which is a byproduct during nanogel synthesis by disulfide bondformation and shows characteristic absorption at 343 nm wavelength; b)Size distribution of nanogels in water; c) TEM images of nanogels (scale500 nm).

FIG. 12. DiI release from nanogels in response to 10 mM GSH over time.

FIG. 13. Top: In vitro cell viability of nanogels on 293T and HeLa cellline after 24 hour of incubation, confocal microscopy images of HeLacells after incubation for 12 hrs with Middle: DiO loaded nanogels(left: DiO channel, middle: DIC image, right: overlap of both) andBottom: FITC conjugated nanogels (left to right: DAPI channel, FITCchannel, overlap of both channels, DIC image with overlap); Scale (20μm).

FIG. 14. Mouse blastocyst formation and embryo transfer results.Blastocysts were formed after 4 days of in vitro culture with (A, A′) orwithout (B, B′) FITC-nanogels. For in vivo experiment, early embryoswere firstly cultured for 3 days in vitro with (C, C′) or without (D)FITC-nanogels, then these morulae/early blastocysts were transferred tothe uteri of recipients. E, live pups born with nanogel exposure duringpreimplantation development.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. General principles of organic chemistry,as well as specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 2006. It will be appreciated that the compounds, as describedherein, may be substituted with any number of substituents or functionalmoieties.

As used herein, “C_(x)-C_(y)” refers in general to groups that have fromx to y (inclusive) carbon atoms. Therefore, for example, C₁-C₆ refers togroups that have 1, 2, 3, 4, 5, or 6 carbon atoms, which encompassC₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, and all likecombinations. “C₁-C₁₅”, “C₁-C₂₀” and the likes similarly encompass thevarious combinations between 1 and 20 (inclusive) carbon atoms, such asC₁-C₆, C₁-C₁₂, C₃-C₁₂ and C₆-C₁₂.

As used herein, the term “alkyl”, refers to a hydrocarbyl group, whichis a saturated hydrocarbon radical having the number of carbon atomsdesignated and includes straight, branched chain, cyclic and polycyclicgroups. The term “hydrocarbyl” refers to any moiety comprising onlyhydrogen and carbon atoms. Hydrocarbyl groups include saturated (e.g.,alkyl groups), unsaturated groups (e.g., alkenes and alkynes), aromaticgroups (e.g., phenyl and naphthyl) and mixtures thereof.

As used herein, the term “C_(x)-C_(y)” alkyl refers to a saturatedlinear or branched free radical consisting essentially of x to y carbonatoms, wherein x is an integer from 1 to about 10 and y is an integerfrom about 2 to about 20. Exemplary C_(x)-C_(y) alkyl groups include“C₁-C₂₀ alkyl,” which refers to a saturated linear or branched freeradical consisting essentially of 1 to 20 carbon atoms and acorresponding number of hydrogen atoms. Exemplary C₁-C₂₀ alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,dodecanyl, etc.

As used herein, the term “halogen” refers to fluorine (F), chlorine(Cl), bromine (Br), or iodine (I).

As used herein, the term “biological, physical or chemicalinterventions” includes a change in pH, redox reagent, redox potential,ionic strength, enzymatic activity, protein concentration, light (e.g.,UVA, UVB or UVC), heat, or mechanical stress.

DESCRIPTION OF THE INVENTION

The invention provides novel polymers and polymeric nanogels havingbiodegradable polymeric backbones, and compositions and methods ofpreparation and use thereof, for example, as guest-host polymernano-assemblies and nano-delivery vehicles, which offer utilities indiverse fields including drug delivery, diagnostics and specialtymaterials.

The polymers, polymeric nanogels and nano-assemblies and nano-deliveryvehicles of the invention can be prepared via simple and reliablesynthetic techniques.

The polymeric nanogels disclosed herein not only exhibit responsivemolecular release, but also show high in vitro cellular viability on HEK293T, MCF7, A549 and HeLa cell lines. The toxicity of these nanogels wasfurther evaluated using mouse preimplantation embryo development as ahighly sensitive toxicity assay, where blastocysts were formed after 4days of in vitro culture and live pups were born when morulae/earlyblastocysts were transferred to the uteri of recipients. These resultsindicate that these nanogels are non-toxic during early mammaliandevelopment and do not alter normal growth.

In one aspect, the invention generally relates to a crosslinkedbiodegradable polymer having a polymeric backbone biodegradable intosmall molecules in response to a relevant biological, physical orchemical stimuli.

In certain preferred embodiments, the polymeric backbone is selectedfrom polyamides, polyesters and polycarbonates.

In certain preferred embodiments, wherein the biodegradable polymercomprises one or more hydrophilic functional groups and one or morecrosslinkable hydrophobic group as part of its side chain.

In another aspect, the invention generally relates to a biodegradablepolymeric nanogel comprising the crosslinked biodegradable polymerdisclosed herein.

In certain preferred embodiments, the biodegradable polymeric nanogelhas encapsulated therein one or more drug or diagnostic payloads,releaseable in response to defined biologically, physically orchemically relevant stimuli. In certain preferred embodiments, whereinthe biologically, physically or chemically relevant stimuli is selectedfrom glutathione concentration inside the cells, a change in pH, redoxreagent, redox potential, ionic strength, enzymatic activity, proteinconcentration, light, heat, and mechanical stress.

In yet another aspect, the invention generally relates to anano-assembly, the nano-assembly includes: a host crosslinked polymernetwork having a biodegradable polymeric backbone, and is surfacefunctionalized with one or more functional groups; and one or more guestmolecular cargos non-covalently encapsulated in the host crosslinkedpolymer network. The host crosslinked polymer network is addressable bya biological, physical or chemical stimuli resulting in partial orcomplete degradation and/or descrosslinking of the host polymer networkand release of the guest molecular cargo from the nano-assembly.

In certain preferred embodiments, the one or more functional groups areadapted to incorporate one or more of small molecule ligands, peptides,proteins, antibodies and aptamers.

In certain preferred embodiments, the biologically, physically orchemically relevant stimuli is selected from glutathione concentrationinside the cells, a change in pH, redox reagent, redox potential, ionicstrength, enzymatic activity, protein concentration, light, heat, andmechanical stress.

In certain preferred embodiments, the biodegradable polymer comprises apolyamide backbone. In certain preferred embodiments, the biodegradablepolymer comprises a polyester backbone. In certain preferredembodiments, the biodegradable polymer comprises a polycarbonatebackbone.

In yet another aspect, the invention generally relates to a method forcontrolled delivery of an agent to a target biological site. The methodincludes: providing a nano-assembly of a host crosslinked polymernetwork non-covalently encapsulating therein a guest molecular cargo,wherein the host crosslinked polymer network is comprised ofbiodegradable polymeric backbones and is adapted to partial or completedecrosslinking and/or degraded by a biological, physical or chemicalintervention resulting in release of the guest molecular cargo from thenano-assembly; delivering the nano-assembly to the target biologicalsite; and causing a biological, physical or chemical interventionresulting in a partial or complete decrosslinking resulting in releaseof the guest molecular cargo from the nano-assembly.

In certain preferred embodiments, the guest molecular cargo is atherapeutic agent. In certain preferred embodiments, the guest molecularcargo is a diagnostic agent. In certain preferred embodiments, the guestmolecular cargo is an imaging agent. In certain preferred embodiments,the guest molecular cargo is a small molecule. In certain preferredembodiments, the guest molecular cargo is a polypeptide. In certainpreferred embodiments, the guest molecular cargo is an oligonucleotide.In certain preferred embodiments, the guest molecular cargo is anantitumor agent.

In certain preferred embodiments, the target biological site comprises asite inside a cell. In certain preferred embodiments, the targetbiological site comprises a site inside a tumor cell. In certainpreferred embodiments, the target biological site comprises a specifictissue. In certain preferred embodiments, the target biological sitecomprises a tumor tissue.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety,    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group, and    -   R is hydrogen or a C₁-C₂₀ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the crosslinked biodegradablepolymer is characterized by a polymeric backbone that comprises:

wherein

-   -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each m is independently an integer from about 1 to about 6        (e.g., 1, 2, 3, 4, 5, 6),    -   each of x and y is independently a positive number, z may be        zero or a positive number,    -   R₁ is a charge-neutral hydrophilic group,    -   R₂ is a group comprising a crosslinking moiety, and    -   R₃ is a non-crosslinking group, selected from a linear or        branched C₁-C₁₆ alkyl group.

In certain preferred embodiments of the above-described polymericbackbones, R₁ is selected from

wherein q is an integer from 3 to about 100 (e.g., from 3 to about 50,from 3 to about 20, from 3 to about 10, from 3 to about 6, from 10 toabout 100, from 20 to about 100),

-   -   a zwitterionic group selected from:

wherein each R is hydrogen or a C₁-C₂₀ alkyl group; n is independentlyan integer from about 1 to about 6 (e.g., 1, 2, 3, 4, 5, 6), or

-   -   a charged functional group selected from —CO₂ ⁻, —HPO₃ ⁻, —SO₃        ⁻, —NR₂ ⁻, —NR₃ ⁺, wherein each R is independently a hydrogen or        a C₁-C₂₀ alkyl group.

In certain preferred embodiments of the above-described polymericbackbones, R₂ comprises at least one stimulus-sensitive functional groupselected from disulfides, acetals, ketals, imines, hydrazides, andoximes.

In yet another aspect, the invention generally relates to a crosslinkedbiodegradable polymer that is biodegradable into molecules that aregenerally regarded as safe (GRAS).

In yet another aspect, the invention generally relates to anano-assembly that is biodegradable into molecules that are generallyregarded as safe (GRAS).

In yet another aspect, the invention generally relates to a method forcontrolled delivery of an agent to a target biological site, wherein thehost crosslinked polymer network is biodegradable into molecules thatare generally regarded as safe (GRAS).

In certain embodiments, the one or more functional groups are adapted toincorporate one or more of small molecule ligands, peptides, proteins,antibodies and aptamers.

In certain preferred embodiments, the biodegradable polymer of theinvention includes a polyester or a polyamide backbone. An exemplarysystem having a PEGylated polyamide backbone having pendent disulfidegroups is shown below.

An exemplary synthetic scheme is provided below. It is understood thatvariations to the synthetic methodologies are available to a personskilled in the art in selecting the suitable approach in preparation ofthe desired polymers.

In certain preferred embodiments, the biodegradable polymer of theinvention includes a polyester backbone. An exemplary system having apolyester backbone is shown below.

In certain preferred embodiments, the biodegradable polymer of theinvention includes a polycarbonate backbone. An exemplary system havinga polycarbonate backbone is shown in FIG. 2.

Exemplary monomers that may be used to form polycarbonates of theinvention include those shown in FIG. 3, which may be synthesizedaccording the following scheme, for example,

Functional cyclic carbonates include, for example, and polymerization offunctional cyclic monomers provide functional polymers.

Exemplary methods for making random copolymers are shown below,including organocatalytic methods from monomers and postfunctionalization of reactive polycarbonates.

In certain embodiments, the polymer network is formed from a homopolymervia a controlled crosslinking. In certain embodiments, the hostcrosslinked polymer network is formed from a random copolymer via acontrolled crosslinking Depending on the nature of the polymer networkand crosslinking, the biological or chemical intervention may be anysuitable event, such as a change in pH, redox reagent, redox potential,ionic strength, enzymatic activity, protein concentration, light, heat,or mechanical stress, which intervention leads to a breaking and/orforming of a chemical bond. For example, certain copolymer-basednanoparticles can be rapidly formed by ultraviolet or visibleirradiation without the need to use any chemical crosslinkers or agents.The encapsulated guest molecules can be released by ultraviolet orvisible irradiation or in the presence of a chemical stimulus such asglutathione if disulfide-bond-forming moieties (or sulfhydryl groups)are also incorporated into the copolymer structure.

In certain embodiments, the one or more functional groups are selectedfrom the group consisting of amino, carboxyl, hydroxyl, halide, acylhalide, ester, azide, nitrile, amide, epoxide, aldehyde, furan, alkeneand alkyne, acyl, and thioacyl groups. In certain preferred embodiments,the one or more functional groups are selected from amino, hydroxyl,amide, halide, carboxyl, acyl, thioacyl and ether groups.

The nano-assembly may take any suitable dimensions, for example, havinga diameter from about 3 nm to about 300 nm (e.g., about 3 nm to about200 nm, about 3 nm to about 100 nm, about 3 nm to about 50 nm, about 3nm to about 30 nm, about 10 nm to about 300 nm, about 30 nm to about 300nm, about 50 nm to about 300 nm, about 100 nm to about 300 nm).

The non-covalently encapsulated guest molecular cargo may be present inany suitable amounts, for example, accounting for from about 1 wt % toabout 45 wt % (e.g., about 1 wt % to about 35 wt %, about 1 wt % toabout 25 wt %, about 1 wt % to about 20 wt %, about 1 wt % to about 15wt %, about 1 wt % to about 10 wt %, about 5 wt % to about 45 wt %,about 10 wt % to about 45 wt %, about 15 wt % to about 45 wt %, about 20wt % to about 45 wt %) of the nano-assembly.

The guest molecular cargo may be any suitable material, for example,selected from therapeutic, diagnostic or imaging agents. For example,the guest molecular cargo is a small molecule, a polypeptide or anoligonucleotide. In certain embodiments, the guest molecular cargo is anantitumor agent. In certain preferred embodiments, the guest molecularcargo is a hydrophobic molecule.

The functionalized surface of the nanogel may display one or morereactive groups at any suitable density, for example, from verysparingly (e.g., about 0.1%) to full coverage (e.g., about 100%). Thus,for example, the functionalized surface of the nanogel may displayreactive groups at a density of, e.g., 1, 2, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, or 100%.

In yet another aspect, the invention generally relates to a method forcontrolled delivery of an agent to a target biological site. The methodincludes: (1) providing a nano-assembly of a host crosslinked polymernetwork non-covalently encapsulating therein a guest molecular cargo,wherein the host crosslinked polymer network is comprised ofbiodegradable polymeric backbones and is adapted to partial or completedecrosslinking and/or degraded by a biological, physical or chemicalintervention resulting in release of the guest molecular cargo from thenano-assembly; (2) delivering the nano-assembly to the target biologicalsite; and (3) causing a biological, physical or chemical interventionresulting in a partial or complete decrosslinking resulting in releaseof the guest molecular cargo from the nano-assembly.

In certain preferred embodiments, the guest molecular cargo is selectedfrom a therapeutic, diagnostic or imaging agent. For example, the guestmolecular cargo is a small molecule, a peptide or an oligonucleotide. Incertain embodiments, the guest molecular cargo is an antitumor agent. Incertain embodiments, the target biological site comprises a site insidea cell (e.g., a tumor cell). In certain embodiments, the targetbiological site comprises a specific tissue (e.g., a tumor tissue). Incertain embodiments, the target biological site comprises a siteextracellular to a tumor cell. In certain embodiments, the nano-assemblyis preferably taken up by a tumor tissue in a physiological environment.

In certain embodiments, the nano-assembly and nanogels of the inventioncomprise polypeptides with side chain functional groups adapted toincorporate side chain functionalities providing for self-assembly andcrosslinking & then decrosslinking based on tissue microenvironment.FIGS. 4-7 illustrate certain exemplary embodiments.

To synthesize a crosslinked polymeric nanogel that contains disulfidecrosslinks using GRAS components, it was assumed that degradation of thepolymeric nanogel under biological conditions will occur throughhydrolysis of esters and amides in addition to the reductive cleavage ofthe disulfide bonds. (Ulery, et al. 2011 J. Polym. Sci., Part B: Polym.Phys. 49, 832-864.) Glutamic acid (a naturally occurring amino acid) andputrescine (one of the growth factors for cell division) were chosen ascomponents of a degradable polyamide backbone. The dicarboxylic acidnature of the glutamic acid and the diamino nature of putrescine wasused to synthesize the amide-based polymer backbone. The amino moiety inthe glutamic acid was then used as the handle to introduce functionalgroups that cause self-assembly of this polymer into a nanogel, as wellas to incorporate surface functional groups, and the crosslinkablefunctional groups. The polyamide, from glutamic acid and putrescine, wasfunctionalized with the hydrophilic oligoethyleneglycol (OEG) moiety andthe hydrophobic pyridyl disulfide (PDS) moiety. Since thisfunctionalization renders the polymer amphiphilic, this is prone toself-assemble, which can then be converted to a crosslinked polymernanogel using the recently introduced self-crosslinking strategy usingthe PDS unit as the handle. (Ryu, et al. 2010 J. Am. Chem. Soc. 132,8246-8247.)

Similarly, in addition to providing the hydrophilic component, the OEGmoiety also has the potential to endow the nanogel with a surfacefunctionality that is known to endow nanocarriers with reducednon-specific interactions in serum. (Peer, et al. 2007 Nat. Nanotechnol.2, 751-760.) Note that the degradation of the side chain functionalgroups in the nanogel will provide oligoethyleneglycol carboxylic acidand thiopropionic acid, both of which are also considered to bebiocompatible and safe. Structures of the targeted polymer and thenanogel are shown in Scheme 2 (FIG. 8).

The precursor polymer was achieved by first synthesizing the polymerbackbone with amino moieties of glutamic acid available for post-polymerfunctionalization. Synthesis of this polymer started with the reactionbetween the bis-N-hydroxysuccinimide ester of N-Boc-L-glutamic acid (1)and putrescine (2) (Scheme 3, FIG. 9). The resultant copolymer, whichwas characterized by ¹H NMR and gel permeation chromatography (GPC), wasfound to have an M_(n) of 8.3 kDa (FIG. S1). Removal of the N-boc-moietyfrom the polymer was achieved using trifluoro acetic acid, theconversion of which was quantitative as discerned by ¹H NMR. The aminein polymer P1 was then treated with excess but equal amounts of theN-hydroxysuccinimide esters of olgioethyleneglycol monocarboxylic acidand PDS-protected thiopropionic acid, as shown in Scheme 3 (FIG. 9).After removing the excess reagents through dialysis, the finalconjugation ratio in the target polymer P2 was determined by ¹H NMR bythe characteristic peaks of the PDS moiety at 8.5 ppm and that of thePEG methoxy group at 3.35 ppm. The ratio of these moieties was found tobe 7:3 for the hydrophilic and hydrophobic side chains. The differencein the conjugation ratio is possibly due to the difference in reactivityof the two side chains.

Since the polymer is amphiphilic in nature, it is expected toself-assemble in aqueous solution. This possibility was evaluated usingdynamic light scattering (DLS) and hydrophobic dye encapsulation studiesin solution, complemented by transmission electron microscopy (TEM)(FIG. 10). DLS experiments were carried out with a 1 mg/mL solution ofpolymer P2 in water and the aggregates were ˜90 nm. Size estimates fromTEM also support the DLS measurement. The TEM data also revealed thatthe assembly has a spherical morphology; the slight departure from aperfectly spherical shape is attributed to the soft nature of thepolymer assembly and the nanogel. Although TEM is the dried version ofthe solution phase assembly, measured in the DLS, since the sizes fromthese two measurements correlate, it is a reasonable assumption that themorphology of the aggregates is indeed spherical in solution. Next testwas whether these amphiphilic polymeric aggregates are capable ofencapsulating hydrophobic molecules in an aqueous medium. To test this,a hydrophobic dye 1,1-dioctadecyl-3,3,3,-tetramethylindocarbocyanineperchlorate (DiI) was used as a fluorescent probe. The dye in itselfdoes not show any absorption in water, as it is not soluble. However,when dissolved in the presence of the polymeric aggregates, its apparentsolubility is evident by absorption spectrum (FIG. 10).

To trap the nanoscale aggregates through chemical crosslinking,self-crosslinking strategy was used in which a sub-stoichiometric amountof dithiothreitol (DTT) is added to the solution containing theseaggregates. Briefly, DTT executes a rapid cleavage of the disulfideunits from the PDS moieties. Since the pyridothione byproduct is stableand unreactive, the thiol moieties generated in the polymer chain nowundergo a thiol-disulfide exchange with the remaining PDS units withinthe aggregate to cause crosslinked polymeric nanogels. A key questionhere is whether this crosslinking reaction is intra-aggregate orinter-aggregate. If this is intra-aggregate, then the size of thecrosslinked nanogel should closely correlate with the amphiphilicaggregate from P2. Indeed, the size of the crosslinked nanogel was foundto be very similar to that of the aggregate (FIG. 11). In generating thenanogels, it is also possible to control the crosslink densities bysimply varying the amount of DTT added to the reaction mixture. To seewhether the extent of crosslinking correlates with the amount of DTTadded, the amount of pyridothione generated in the reaction wasmonitored, which was found to closely correlate with each other. Thissuggests that the DTT-induced cleavage of the PDS units and thesubsequent crosslinking reactions are nearly quantitative. Two nanogels,NG1 and NG2 with 3% and 5% cross-linking densities, respectively, wereprepared. NG2 was used for all the subsequent studies outlined below.

Once a molecule is encapsulated within the interior, it is also criticalto be able to trigger the release of these molecules in response to aspecific stimulus. Since the nanogels consist of disulfide crosslinks,they should be responsive to thiol-based reducing environments.Glutathione (GSH) is a reducing agent found in millimolar concentrationsin the cytosol, while its concentration in the extracellular environmentis micromolar. Thus, the release of the encapsulated dye molecule fromthe NG2 nanogel scaffold was tested in the presence of 10 mM GSH. It wasobserved that in the presence of GSH, the guest molecule was releasedfrom the nanogel as discerned from the decrease in the absorptionspectrum. The decrease in absorbance of DiI is attributed to theprecipitation of the rather hydrophobic guest molecule in water, uponrelease from the nanogel due to the GSH-triggered decrosslinking (FIG.12). In a control experiment, where no GSH was added to the solution,the decrease in absorbance was much slower which indicates that theguest molecule release is indeed occurring due to the decrosslinking ofthe nanogel scaffold in the presence of GSH.

A key motivation behind designing a polymer with GRAS components is todesign a polymeric nanogel that exhibits very low toxicity. First testedwas the in vitro cellular viability of the nanogels on HEK-293T, MCF7,A549 and HeLa cell lines using the Alamar blue assay. NG2, which wasincubated along with cell lines for 24 hours at 37° C., showed high andconcentration independent cellular viability for up to a concentrationof 0.5 mg/mL (FIG. 13).

Cytotoxicity studies are meaningful, only when the nanoassembly gainsaccess to the cells and still prove not to be cytotoxic. Therefore, itwas also tested whether these nanogels can undergo cellularinternalization. In vitro cellular uptake of nanogels encapsulated withhydrophobic dye, 3,3′-dioctadecyloxa-carbocyanine perchlorate (DiO) wereperformed, where the nanogels were incubated with HeLa cells for 12hours and evaluated by confocal microscopy. It was noted that nanogelsenter the cells within this time period and that these are distributedthroughout the cytoplasm, as shown in FIG. 13. It is also possible thatthe guest molecules can leak from the nanogels, where the hydrophobicdyes can passively diffuse into the cells. If DiO escaped the nanogel,it would mainly bind to the cell membrane rather than diffuse into thecytosol.

To confirm that the DiO signal observed was not due to escape fromnanogels, fluorescein was covalently attached to the nanogels andexamined them for cellular uptake. The fluorescein-labeled nanogels weresimilarly incubated with HeLa cells for 12 hours at 37° C. and examinedby confocal microscopy. It is again clear that the nanogels were notonly taken up by the cells, but also are uniformly distributedthroughout the cytosol (FIG. 13).

The toxicity of the nanogels was further evaluated using a more rigoroustest. Mouse preimplantation embryos were cultured in the presence ofnanogels. Preimplantation embryos are generally more sensitive totoxicants than regular somatic cells and must undergo severalmorphogenetic events in order to successfully develop into a blastocystover a 4-day period. Perturbations in many cellular events, defectivecell cycle and cell lethality are known to disrupt blastocystdevelopment. (Lin, et al. 2009 Hum. Reprod. 24, 386-397; Taylor, et al.2014 Beilstein J. Nanotechnol. 5, 677-688; Fleming, et al. 2004 BiolReprod. 71, 1046-1054.) Therefore, assessing the development of embryosin the presence of nanogels is a highly sensitive method for evaluatingtoxicity.

The development of embryosin culture, with and without nanogels, wascarefully monitored. No differences were found in development rate orefficiency of blastocyst formation in the presence of nanogels. Both thecontrol group (KSOM, negative control) and KSOM supplemented withfluorescein-labeled nanogels (nanogel-FITC) developed blastocysts after4 days in culture (FIG. 13A-B). In addition, fluorescence signal wasalso detected in nanogel-FITC blastocysts, but not in the negativecontrols (FIG. 14A-B) indicating that the nanogel-FITC was taken up byblastomeres during preimplantation development, but did not exhibit anydisruption to developmental potential of embryos. These results showthat the nanogels are highly biocompatible and non-toxic to mammalianpreimplantation embryos and pluripotent cells.

To further investigate toxicity, early blastocysts were transferred intopseudopregnant recipients in order to determine pups can be born after 3days of preimplantation nanogel exposure. Live pups were born atequivalent rates to controls after preimplantation development in thepresence of glutamic acid nanogels (FIG. 14D). Taken together, theseresults indicate that the glutamic acid nanogels are biocompatible andnon-toxic to mammalian preimplantation development, which is quitesensitive to the culture environment and that fetal development afterculture in the nanogels is similarly unaffected permitting normal growthand survival to birth. (Watkins, et al. 2008 Semin. Reprod. Med. 26,175-185.)

Thus, a new polyamide has been developed into a nanogel with thebuilding blocks that are based on biocompatible components. The backboneof the polyamide is based on glutamic acid and putrescine, while theside chain substituents are based on oligoethyleneglycol andthiopropionic acid. Since the precursor to these side-chain substituentsmake the polyamide amphiphilic, the polymer self-assembles in aqueoussolutions. Disulfide crosslinked polymeric nanogels have been obtainedusing this self-assembly, while concurrently taking advantage of theamphiphilic nature of the assembly to sequester hydrophobic moleculeswithin its interior. Since the crosslinks are based on disulfidefunctionalities, these nanogels exhibit molecular release in response tothe intracellular stimulus, glutathione. Finally, to test theversatility of the biocompatible nanogel design, these nanogels weretested for toxicity using a more classical cytotoxicity assay and a morerigorous and highly sensitive mammalian preimplantation developmentassay. In both assays, the nanogels exhibit no discernible toxicity,suggesting that these GRAS-based stimuli responsive nanogels has greatpotential for in vivo applications.

Experimental Materials

All the reagents were purchased from commercial source and used as suchwithout further purification, unless otherwise mentioned. ¹H NMR spectrawere recorded on a Bruker DPX-400 MHz NMR spectrometer and all thespectra were calibrated against tetramethylsilane (TMS). Dynamic LightScattering (DLS) measurements were carried out on a MalvernNanozetasizer. TEM images were recorded on a JEOL-2000FX machineoperating at an accelerating voltage of 100 kV cell imaging wasperformed using a Zeiss 510 META confocal microscope. Fluorescencemeasurements were performed on a fluorescence plate reader (MolecularDevices, SpectraMax M5).

Synthesis of Polymer 1b:

100 mg (0.226 mmol) of N-boc-L-glutamic acid di-activated ester wasadded in a round bottom flask with DMF for stirring. 52.47 μL (0.376mmol) of triethyl amine was then added to the solution followed byadding 22.7 μL (0.226 mmol) of 1,4 diaminobutane. The reaction wasstirred overnight and quenched by cooling it. The final product waspurified by precipitating it in diethyl ether followed by dialysis usinga cut off membrane of Mn 3.5 kDa in methanol. The product is isolated asan off-white sticky solid. Finally, polymer 1 is obtained by TFAdeprotection.

Synthesis of Nanogel Precursor Polymer P2:

75 mg (0.3483 mmol) of polymer 1 and 974 (0.6966 mmol) of triethyl aminewas dissolved in DMF in a round bottom flask. 113 mg (0.2786 mmol) ofPEG-NHS 3 and 87 mg (0.2786 mmol) of PDS-NHS 2 was then added to thereaction mixture and stirred overnight. The product was then dialyzed inmethanol with a 3.5 kDa cutoff membrane.

Procedure for Dye Encapsulation:

The polymer P2 (1 mg/mL) was dissolved in water. 10 μL (0.01 mg) of DiI(1 mg/mL in acetone) was added to the stirring solution of polymerfollowed by the desired amount of DTT for crosslinking. The mixture wasstirred overnight at room temperature, open to the atmosphere allowingthe organic solvent to evaporate. Excess insoluble DiI was removed byfiltration and pyridothione was removed from the nanogel solution bydialysis using a membrane with molecular cut-off of 3.5 kDa

Dynamic Light Scattering (DLS) Study:

For the DLS measurements, the concentration of the polymer and nanogelsolution was 1 mg/mL. The solution was filtered using a hydrophilicmembrane (pore size 0.450 μm) before experiment was performed.

Transmission Electron Microscope (TEM) Study:

For the TEM measurements the nanogel solution was prepared in 1 mg/mLconcentration. One drop of the sample was dropcasted on carbon coated Cugrid. About 3 min after the deposition, the grid was tapped with filterpaper to remove surface water. Finally, it was dried in air for another6 h before images were taken.

In Vitro Cell Viability:

The in vitro cellular viability of the nanogels was evaluated on healthyHEK293T, MCF7, A549, and HeLa cancer cell lines. The cells were culturedin T75 cell culture flasks using Dulbecco's Modified EagleMedium/Nutrient Mixture F-12 (DMEM/F12) with 10% fetal bovine serum(FBS) supplement. The cells were seeded at 10,000 cells/well/200 μL in a96 well plate and allowed to grow for 24 hours under incubation at 37°C. and 5% CO₂. These cells were then treated with nanogels of differentconcentrations and were incubated for another 24 hours. Cell viabilitywas measured using the Alamar Blue assay with each data point measuredin triplicate. Fluorescence measurements were made using the platereader SpectraMax M5 by setting the excitation wavelength at 560 nm andmonitoring emission at 590 nm on a black well plate.

In Vitro Cell Uptake:

FITC labeled nanogels was synthesized using the amines in the polymerfor conjugation with FITC. In a vial, polymer was dissolved in methanoland excess FITC was added. The solution was let to stir overnightfollowed by extensive dialysis in methanol using cut off membraneM_(n)-3500 Da. Nanogel was prepared using the same procedure asdescribed previously.

In a glass bottom dish, HeLa cells were incubated overnight at 37° C.and 5% CO₂ with nutrient medium (DMEM/F12 with 10% fetal bovine serumsupplement). The nutrient medium was then taken out and the cells werewashed with pH 7.4 PBS buffer. To it 100 μL of the nanogel solution (10mg/mL) either encapsulated with DiO or conjugated with FITC were addedalong with the nutrient medium. The cells were then incubated for 30 minat 37° C. and the fluorescence was observed under a confocal microscope(63× oil immersion objective)

Embryo Recovery and Culture:

B6D2F1 female mice (8 to 10 weeks old) were induced to superovulate with5 IU pregnant mare's serum gonadotropin (PMSG, Sigma), followed 46-48 hlater by 5 IU human chorionic gonadotropin (hCG, Sigma). Females weremated with B6D2F1 males immediately after hCG injection, and euthanizedat 20-22 h post-hCG injection. Oviductal ampullae were cut open torelease zygotes, and cumulus cells were removed by pipetting in M2medium (Millipore) containing 0.1% hyaluronidase (Sigma). Zygotes werecultured in KSOM medium (Millipore) or KSOM supplemented with 1 mg/mL ofnanogel solution at 37° C., 5% CO₂/5% O2 balanced in N2 for 4 days. Useof vertebrate animals for embryo production was approved by theUniversity of Massachusetts IACUC.

Embryo Transfer:

Morulae/early blastocysts after 3 days of culture in KSOM or KSOMsupplemented with nanogels were transferred into uteri of 2.5 dpc (daypost coitus) pseudopregnant foster dams (CD-1 mice, albino) by using thenon-surgical embryo transfer (NSET) device. Recipient females wereallowed to deliver pups naturally in order to observe production of livehealthy animals after preimplantation development in the presence ofnanogel solution.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood too one of ordinary skill in the art. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art.Methods recited herein may be carried out in any order that is logicallypossible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

1. A crosslinked biodegradable polymer comprising a polymeric backbonebiodegradable into small molecules in response to a relevant biological,physical or chemical stimuli.
 2. The crosslinked biodegradable polymerof claim 1, wherein the polymeric backbone is selected from polyamides,polyesters and polycarbonates and is biodegradable into molecules thatare generally regarded as safe (GRAS).
 3. The crosslinked biodegradablepolymer of claim 2, wherein the biodegradable polymer comprises one ormore hydrophilic functional groups and one or more crosslinkablehydrophobic group as part of its side chain.
 4. A biodegradablepolymeric nanogel comprising the crosslinked biodegradable polymer ofclaim
 1. 5. The biodegradable polymeric nanogel of claim 4, havingencapsulated therein one or more drug or diagnostic payloads,releaseable in response to defined biologically, physically orchemically relevant stimuli.
 6. The biodegradable polymeric nanogel ofclaim 5, wherein the biologically, physically or chemically relevantstimuli is selected from glutathione concentration inside the cells, achange in pH, redox reagent, redox potential, ionic strength, enzymaticactivity, protein concentration, light, heat, and mechanical stress. 7.A nano-assembly comprising: a host crosslinked polymer network having abiodegradable polymeric backbone, and is surface functionalized with oneor more functional groups; and one or more guest molecular cargosnon-covalently encapsulated in the host crosslinked polymer network;wherein the host crosslinked polymer network is addressable by abiological, physical or chemical stimuli resulting in partial orcomplete degradation and/or descrosslinking of the host polymer networkand release of the guest molecular cargo from the nano-assembly.
 8. Thenano-assembly of claim 7, wherein the one or more functional groups areadapted to incorporate one or more of small molecule ligands, peptides,proteins, antibodies and aptamers, wherein the one or more functionalgroups are selected from amino, hydroxyl, amide, halide, carboxyl, acyl,thioacyl and ether groups.
 9. (canceled)
 10. The nano-assembly of claim7, wherein the biologically, physically or chemically relevant stimuliis selected from glutathione concentration inside the cells, a change inpH, redox reagent, redox potential, ionic strength, enzymatic activity,protein concentration, light, heat, and mechanical stress.
 11. Thenano-assembly of claim 7, wherein the biodegradable polymer comprises apolyamide backbone, a polyester backbone, or a polycarbonate backboneand is biodegradable into molecules that are generally regarded as safe(GRAS).
 12. (canceled)
 13. (canceled)
 14. The crosslinked biodegradablepolymer of claim 1, wherein the polymeric backbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachof x and y is independently a positive number, z may be zero or apositive number, R₁ is a charge-neutral hydrophilic group, R₂ is a groupcomprising a crosslinking moiety, and R₃ is a non-crosslinking group,selected from a linear or branched C₁-C₁₆ alkyl group.
 15. Thecrosslinked biodegradable polymer of claim 1, wherein the polymericbackbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachof x and y is independently a positive number, z may be zero or apositive number, R₁ is a charge-neutral hydrophilic group, R₂ is a groupcomprising a crosslinking moiety, and R₃ is a non-crosslinking group,selected from a linear or branched C₁-C₁₆ alkyl group.
 16. Thecrosslinked biodegradable polymer of claim 1, wherein the polymericbackbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachm is independently an integer from about 1 to about 6, each of x and yis independently a positive number, z may be zero or a positive number,R₁ is a charge-neutral hydrophilic group, R₂ is a group comprising acrosslinking moiety, and R₃ is a non-crosslinking group, selected from alinear or branched C₁-C₁₆ alkyl group.
 17. The crosslinked biodegradablepolymer of claim 1, wherein the polymeric backbone comprises:

wherein each of x and y is independently a positive number, z may bezero or a positive number, R₁ is a charge-neutral hydrophilic group, R₂is a group comprising a crosslinking moiety, R₃ is a non-crosslinkinggroup, selected from a linear or branched C₁-C₁₆ alkyl group, and R ishydrogen or a C₁-C₂₀ alkyl group.
 18. The crosslinked biodegradablepolymer of claim 1, wherein the polymeric backbone comprises:

wherein each of x and y is independently a positive number, z may bezero or a positive number, R₁ is a charge-neutral hydrophilic group, R₂is a group comprising a crosslinking moiety, and R₃ is anon-crosslinking group, selected from a linear or branched C₁-C₁₆ alkylgroup.
 19. The crosslinked biodegradable polymer of claim 1, wherein thepolymeric backbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachof x and y is independently a positive number, z may be zero or apositive number, R₁ is a charge-neutral hydrophilic group, R₂ is a groupcomprising a crosslinking moiety, and R₃ is a non-crosslinking group,selected from a linear or branched C₁-C₁₆ alkyl group.
 20. Thecrosslinked biodegradable polymer of claim 1, wherein the polymericbackbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachof x and y is independently a positive number, z may be zero or apositive number, R₁ is a charge-neutral hydrophilic group, R₂ is a groupcomprising a crosslinking moiety, and R₃ is a non-crosslinking group,selected from a linear or branched C₁-C₁₆ alkyl group.
 21. Thecrosslinked biodegradable polymer of claim 1, wherein the polymericbackbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachof x and y is independently a positive number, z may be zero or apositive number, R₁ is a charge-neutral hydrophilic group, R₂ is a groupcomprising a crosslinking moiety, and R₃ is a non-crosslinking group,selected from a linear or branched C₁-C₁₆ alkyl group.
 22. Thecrosslinked biodegradable polymer of claim 1, wherein the polymericbackbone comprises:

wherein each m is independently an integer from about 1 to about 6, eachm is independently an integer from about 1 to about 6, each of x and yis independently a positive number, z may be zero or a positive number,R₁ is a charge-neutral hydrophilic group, R₂ is a group comprising acrosslinking moiety, and R₃ is a non-crosslinking group, selected from alinear or branched C₁-C₁₆ alkyl group. 23-26. (canceled)
 27. A methodfor controlled delivery of an agent to a target biological site,comprising: providing a nano-assembly of a host crosslinked polymernetwork non-covalently encapsulating therein a guest molecular cargo,wherein the host crosslinked polymer network is comprised ofbiodegradable polymeric backbones and is adapted to partial or completedecrosslinking and/or degraded by a biological, physical or chemicalintervention resulting in release of the guest molecular cargo from thenano-assembly; delivering the nano-assembly to the target biologicalsite; and causing a biological, physical or chemical interventionresulting in a partial or complete decrosslinking resulting in releaseof the guest molecular cargo from the nano-assembly. 28-52. (canceled)